The present invention relates to labeled compounds and more particularly to compounds labeled with carbon-13 and hydrogen-2.
Stable isotope labeled amino acids and nucleotides are required for structural and mechanistic studies of proteins and oligonucleotides. In addition, isotopically labeled biologically active compounds are required for many phases of drug discovery and development including elucidation of biosynthetic pathways, pharmacokinetics, and drug metabolism. For many applications, site-specific 13C or combined 13C and 2H labeling are required. While a number of stable isotope labeled compounds are available from companies such as Sigma-Aldrich Chemicals, a need remains for other labeled synthetic precursors.
Methyl aryl sulfides such as methyl phenyl sulfide have been used in a wide number of reactions to make a large number of such biomolecules and other important synthetic precursors. For example, methyl phenyl sulfide can be used as a nucleophilic synthon and is easily converted into an electrophilic synthon. While methyl phenyl sulfide could provide a chemically stable and non-volatile carrier for the valuable 13C and 2H labels, the preparation of few isotopically labeled methyl phenyl sulfides has been previously accomplished. One example of an isotopically labeled methyl phenyl sulfide labeled at the methyl group is shown by Chaudhary et al., J. Labelled Cpd. Radiopharm., 43, 683-691 (2000), although that sulfide wasn""t described or suggested as a synthetic reagent for synthesis of labeled compounds. Availability of other significant [2H1, 13C], [2H2, 13C] and [2H3, 13C]methyl phenyl sulfides would allow researchers to take advantage of the wealth of chemistry that has been done using unlabeled methyl phenyl sulfide.
As carbon-13 is separated from its lighter isotope by cyrogenic distillation of carbon monoxide (CO), all labeled carbons are derived ultimately from CO. The highly efficient conversion of CO to useful chemical precursors is perhaps the most unique aspect of stable isotope labeling technology. Any inefficiencies in the early synthetic steps add greatly to the overall expense of isotope labeling. Thus, considerable efforts have been directed to the development of methods for the preparation of useful synthetic precursors or synthons. This effort has given rise to efficient large-scale methods for the synthesis of methane, methanol, methyl iodide, sodium formate, potassium cyanide and carbon dioxide. These methods are the foundation of all labeling chemistry. The most useful of the electrophilic one-carbon precursors, methyl iodide and carbon dioxide, are difficult to store and use efficiently due to their high volatility.
As spectroscopic instrumentation and techniques continue to improve, there is a drive to study ever more complicated bio-systems. This has lead to demands for more complex labeling patterns in biomolecules. In the past, the simple introduction of a labeled atom site-specifically without stereospecificity was the major thrust for stable isotope labeling and the first generation of labeled synthons served this effort well. Increasingly, in today""s labeling climate, in addition to site-specific labeling, the requirement for stereospecificity has been added. This includes both the ability to stereospecific label chiral compounds as well as the ability to differentiate between prochiral centers with deuterium or carbon. Additional synthons as starting materials will address those growing demands.
It is an object of the present invention to provide labeled compounds.
In accordance with the purposes of the present invention, as embodied and broadly described herein, the present invention provides labeled compounds, [2H1, 13C], [2H2, 13C] and [2H3, 13C]methyl aryl sulfides wherein the 13C methyl group attached to the sulfur of the sulfide includes exactly one, two or three deuterium atoms and the aryl group is selected from the group consisting of 1-naphthyl, substituted 1-naphthyl, 2-naphthyl, substituted 2-naphthyl, and phenyl groups with the structure 
wherein R1, R2, R3, R4 and R5 are each independently, hydrogen, a C1-C4 lower alkyl, a halogen, an amino group from the group consisting of NH2, NHR and NRRxe2x80x2 where R and Rxe2x80x2 are each a C1-C4 lower alkyl, a phenyl, or an alkoxy group.
The present invention further provides a process of preparing a [2H1, 13C], [2H2, 13C] and [2H3, 13C]methyl aryl sulfide wherein the 13C methyl group attached to the sulfur of the sulfide includes exactly one, two or three deuterium atoms and the aryl group is selected from the group consisting of 1-naphthyl, substituted 1-naphthyl, 2-naphthyl, substituted 2-naphthyl, and phenyl groups with the structure 
wherein R1, R2, R3, R4 and R5 are each independently, hydrogen, a C1-C4 lower alkyl, a halogen, an amino group from the group consisting of NH2, NHR and NRRxe2x80x2 where R and Rxe2x80x2 are each a C1-C4 lower alkyl, a phenyl, or an alkoxy group, the process including reacting [13C]methanol with hydriodic acid to form a product mixture including volatile [13C]methyl iodide, passing said volatile [13C]methyl iodide directly into a biphasic mixture including aqueous sodium hydroxide, benzene, and an arylthiol having the structure 
wherein R1, R2, R3, R4 and R5 are each independently a C1-C4 lower alkyl, a halogen, an amino group from the group consisting of NH2, NHR or NRRxe2x80x2 where R and Rxe2x80x2 are each a C1-C4 lower alkyl, a phenyl or an alkoxy group and maintaining said biphasic mixture for time sufficient to form the, [2H1, 13C], [2H2, 13C] and [2H3, 13C]methyl aryl sulfide
Methyl aryl sulfides are useful organic reagents that allows for the construction of many useful biochemicals and materials. Isotopically labeled methyl aryl sulfide can be used to introduce a carbon-13 [13C] and a hydrogen-2 or deuterium label [2H] into such biochemicals and materials.
As used herein, the term xe2x80x9carylxe2x80x9d means a monovalent monocyclic or bicyclic aromatic hydrocarbon radical of 6 to 10 ring atoms, and optionally substituted independently with one, two, three, four or five substituents selected from alkyl, haloalkyl, cycloalkyl, halo, nitro, cyano, xe2x80x94OR (where R is hydrogen, alkyl, haloalkyl, cycloalkyl, optionally substituted phenyl), acyl, and xe2x80x94COOR (where R is hydrogen or alkyl). More specifically, the term xe2x80x9carylxe2x80x9d includes, but is not limited to 1-naphthyl, substituted 1-naphthyl, 2-naphthyl, substituted 2-naphthyl, and phenyl groups with the structure 
wherein R1, R2, R3, R4 and R5 are each independently a lower alkyl, i.e., a C1-C4 alkyl such as methyl, ethyl, n-propyl, iso-propyl, butyl, isobutyl, and tert-butyl, a halogen such as chloro, bromo or iodo, an amino group such as NH2, NHR or NRRxe2x80x2 where R and Rxe2x80x2 are each a lower alkyl or aryl as described above, or an alkoxy group such as O-alkyl or O-aryl where the alkyl is a lower alkyl as described above or an aryl as described above.
As used herein, the term xe2x80x9c[2H1, 13C]xe2x80x9d means exactly one deuterium atom, the term xe2x80x9c[2H2,13C]xe2x80x9d means exactly two deuterium atoms, and the term xe2x80x9c[2H3, 13C]xe2x80x9d means exactly three deuterium atoms within the respective compound.
[2H1, 13C], [2H2,13C] and [2H3,13C]Methyl aryl sulfides can be made from [2H1, 13C], [2H2, 13C] and [2H3, 13C]methyl alcohol in a one step process as shown below. The [2H1, 13C], [2H2, 13C] and [2H3, 13C]methyl aryl sulfides can be used as a non-volatile carrier of the desired labels, i.e., carbon and hydrogen labels. Methyl aryl sulfides without the isotopic substitution can be made in high yields by the same process. 
Availability of the [2H1, 13C], [2H2, 13C] and [2H3, 13C]methyl aryl sulfides having the structure 
wherein R1, R2, R3, R4 and R5 are each independently a lower alkyl, i.e., a C1-C4 alkyl such as methyl, ethyl, n-propyl, iso-propyl, butyl, isobutyl, and tert-butyl, a halogen such as chloro, bromo or iodo, an amino group such as NH2, NHR or NRRxe2x80x2 where R and Rxe2x80x2 are each a lower alkyl or aryl as described above, or an alkoxy group such as O-alkyl or O-aryl where the alkyl is a lower alkyl as described above or an aryl as described above, and the methyl group attached to the sulfide includes exactly one, two or three deuterium atoms will allow researchers to take advantage of the wealth of chemistry that has been done using unlabeled methyl phenyl sulfide.
The present invention provides [2H1, 13C], [2H2, 13C] and [2H3, 13C]methyl aryl sulfides, i.e., [13C]methyl aryl sulfide wherein the methyl group includes exactly one, two or three deuterium atoms. For those molecules with exactly one or two deuterium atoms, such isotopically differentiated methyl groups can be attractive for a variety of applications. For example, a chirally differentiated isopropyl group, i.e., xe2x80x94CH(13CDH2)(13CD2H) can be produced.
The present invention provides efficient large scale one-pot processes for the preparation of methyl phenyl sulfides, e.g., [2H1,13C], [2H2, 13C] and [2H3, 13C]methyl phenyl sulfides from [2H1, 13C], [2H2, 13C] and [2H3, 13C]methanol. Such processes can avoid the inevitable losses resulting from the isolation of labeled methyl iodide. Such methyl aryl sulfides provide a chemically stable and non-volatile carrier for the valuable 13C and 2H labels.
In addition, [13C]methyl iodide including exactly one or two deuterons has been prepared from [2H1, 13C] and [2H2, 13C]methyl aryl sulfides including exactly one or two deuterons. Such materials, i.e., [2H1, 13C] and [2H2, 13C]methyl iodides have not previously been available.
[13C]Methanol is commercially available. Thus, labeled methyl iodide could be derived from the readily available labeled methanol by refluxing the methanol in hydriodic acid (47-57% by weight in water) and collecting the volatile components. The resultant material can then be dried by treatment with a molecular sieve and re-distilled to yield [13C]methyl iodide in yields varying from 62% to 94% according to prior publications. Unfortunately, this easy synthesis suffers a drawback and potential hazard from the fact that methyl iodide is both volatile and carcinogenic. To avoid these problems, the process of the present invention combines two synthetic steps into a single pot process and avoids the isolation of [13C]methyl iodide.
In the process of the present invention, [13C]methyl aryl sulfides can be prepared in a high yield ( greater than 95%) process by refluxing [13C]methanol in hydriodic acid (47% by weight in water) to produce volatile methyl iodide, and passing the volatile methyl iodide into a biphasic mixture of thiophenol, sodium hydroxide, water and benzene for time sufficient to form [13C]methyl phenyl sulfide.
The [13C, 2H1]methyl iodide and [13C, 2H2]methyl iodide can be prepared from [13C, 2H1]methyl phenyl sulfide and [13C, 2H2]methyl phenyl sulfide. Initially, quantitative deprotonation of either [13C]methyl phenyl sulfide or [13C, 2H3]methyl phenyl sulfide can be accomplished by reaction with sec-butyllithium at xe2x88x9278xc2x0 C. Use of sec-butyllithium is preferred over n-butyllithium because the need for co-solvents and other additives can be eliminated. Based on NMR analysis, the deuterium content of the product sulfides was equivalent to the deuterated starting materials. Finally, the labeled methyl iodides are formed in good yield by slowly heating benzyl iodide and the respective methyl phenyl sulfide to around 160xc2x0 C. and collecting the volatile methyl iodide products using a liquid nitrogen cooled trap.
The present invention is more particularly described in the following examples which are intended as illustrative only, since numerous modifications and variations will be apparent to those skilled in the art.